猪源大肠杆菌O_(157)∶H_7菌体蛋白双向电泳方法的建立

Establishment of two-dimensional electrophoresis assay for proteome analysis of Escherichia coli O_(157)∶H_7

为了建立大肠杆菌O157∶H7菌体蛋白的双向电泳技术,获得背景清晰,蛋白点分辨率高,重复性好的双向电泳图谱。对双向电泳技术中的样品制备方法、上样量、染色等关键步骤进行了优化。最终选用冰浴超声(9s/间隔9s,200w)3min,再置4℃24h后离心提取蛋白样品,以上样量为0.5mg菌体蛋白在pH值4-7的24cm IPG胶条上进行电泳,最后用考马斯亮蓝G-250染色,获得了较好的双向电泳图谱,得到了850±20个蛋白质点,蛋白主要集中在pH值4.06-7.53之间,匹配率为95.3%。随机选取10个蛋白质点进行质谱鉴定,检出率100%,10个蛋白质除了一个蛋白质为假定蛋白以外,其他蛋白质分属于乙酰辅酶A合成、能量代谢、氨基酸代谢等代谢途径。建立的大肠杆菌O157∶H7菌体蛋白双向电泳方法,为开展大肠杆菌O157∶H7强弱毒力菌株的差异蛋白质组学研究奠定了基础。

To establish a two-dimensional electrophoresis （2-DE） assay for proteome analysis of Esche- richia coli O157 ： H7 and to get an electrophoretogram with a clear background, high protein point resolu- tion and good repeatability, 2-DE program was optimized, and various sample preparation methods, differ- ent loading quantities and different dyeing methods were studied. Samples were ultra sounded with ice bath at 3 minute （9 s/every 9 s） followed by deposited at 4C at 24 hours. 0.5 mg total protein were loaded onto pH4-7 IPG strip for 2-DE followed by staining with Coomassie Brilliant Blue G-250 nitrate. 2-DE image a- nalysis indicated that 850 4-20 protein spots were recognized, and protein spots were mainly focused be- tween pI values 4.06 to 7.53. Matching rate of repeated electrophoregrams was 95.3%. Ten proteins were randomly chosen to identify by ma ~pectrum, and detection rate was 100%. Ten proteins were involved in synthesis of Con A, energy metabolism, amino acids metabolism, etc except one protein was hypothet- ical protein. Establishment of 2-DE assay for Escherichia coli O157 ： H7 in this study provide the founda- tion for Escherichia coli O157： H7 proteomics research.